1. Project Summary/Abstract. Crohn's disease (CD) is a chronic inflammatory bowel disease (IBD) that affects millions of people in a progressive manner. Despite great advancements in therapy, it remains unclear why inflammation in the digestive system is progressive and leads to complications (e.g., enteric fistulas) that are only alleviated by surgical removal of damaged bowel. Over the next decade, thousands of patients will have surgery in the USA, but as a medical problem, most (<88%) will have CD reoccurrence over time. Concerningly, CD recurs at the anastomosis site where the bowel-ends are reconnected, but intriguingly, of all therapies, antimicrobial metronidazole is the only one that slows recurrence, indicating that bacteria could play a harmful role in CD. We recently developed a uniquely rapid, real-time stereomicroscopic (SM) profiling strategy of fresh surgical specimens, and discovered a novel system of 3D-microscopic cavernous fistulous tract lesions (CavFT) in the bowel of CD patients that underwent surgery. Of notice, CavFTs were unnoticed by standard pathology/surgical methods. More recently, our studies showed that such CavFT lesions spread microscopically within the gut wall, and that contain bacterial species that could be contributors to the severe complications observed in CD patients. CavFTs could also explain postoperative CD recurrence. Together, the findings provide the strong scientific premise to indicate that CavFTs are collectively a microscopic medical and microbiological problem that remains uncharacterized in CD. The central hypothesis in this proposal is that the bowel of surgical CD patients has CavFT pathogenic bacteria that aggravate CD and the severity of its complications. The main objective of this proposal is to experimentally determine if CavFT bacteria play a pathogenic role in CD using a unique mouse model of CD-like ileitis (SAMP1/YitFc), and phagocytosis assays. Herein, we propose three aims that complementary will characterize the correlation between the magnetic resonance imaging ex vivo analysis (exMRI) of surgically removed CD bowels and that of the bacteria that thrive in such pathogenic CavFTs. Prevalent cultivable CavFT bacteria will be administered to SAMP1/YitFc mice to identify global mechanisms of intestinal disease intensification in CD. Since CD primarily affects the ileum, this proposal is clinically relevant because SAMP1/YitFc is the most advanced animal model of spontaneous CD-like ileitis to test our hypothesis. If our assumption is correct, the identification of pathogenic bacteria thriving inside microscopic CavFT indicates that adapted bacteria that evade immune clearance could become easy targets for routine diagnostics and therapy in perioperative CD patients. As deliverables, this proposal will provide i) a list of proinflammatory bacteria, with rates of isolation and antiphagocytic activity, and ii) a list of proinflammatory pathways that could restrain the evolution of CavFT lesions. This proposal is significant because it will provide i) scientific evidence about the bacterial causes of CavFT, and ii) novel insights on how to deliver effective drugs into fistulous-space lesions to promote healing. 1